Background: Hepatocellular carcinoma (HCC) arises in an inflammatory, hypoxic/acidic microenvironment that favours tumour progression and fosters immunosuppression. Tumour cells survive this hostile environment by over-expressing pH regulatory molecules such as carbonic anhydrase (CA) IX, XII and V-ATPase complex, but the relevance of these molecules in HCC is poorly defined.
Aim: The aim of this study was to dissect the relationships between pH regulatory molecules and the aggressive behaviour of malignant hepatocytes, and to evaluate how pH regulatory molecules influence the immune microenvironment of HCC.
Methods: HCC, non-tumour and normal liver tissue samples were analysed by qRT-PCR for the expression of genes encoding the pH regulatory molecules (CAIX, CAXII and V-ATPase), of genes associated to epithelial-to-mesenchymal-transition (EMT) (TWIST, CDH1, VIM) and those encoding for HCC stem cell-associated markers (CD13, CD24, CD44, CD90, EpCAM, CD133, KRT19, OCT4, NANOG and SOX2). Selected HCC, non-tumour and normal liver tissue samples were evaluated by immunohistochemistry (IHC) to detect the presence and localization of CAIX, CAXII and VATPase and to assess the distribution of macrophages and T cells. Confocal microscopy and flow cytometry were implemented to assess the co-expression of selected markers. HCC cell lines, characterised for the expression of pH regulators, were tested for the sensitivity to the CAIX, CAXII, and V-ATPase specific inhibitors. The effects of V-ATPase specific drug were also studied ex vivo in primary human HCC tumour explants by qRT-PCR and by flow cytometry in HCC single cell suspensions obtained by the enzymatic digestion of HCC specimens.
Results: Our mRNA analysis showed that the expression of CA9 was significantly correlated with the expression of the hypoxia-inducible factor 1α-related gene (HIF1A) and of the stem cell-associated markers CD24, CD133, EpCAM and KRT19. Moreover, mRNA for CA9 and for the different CA12 isoforms were associated with tumour grading, thus indicating their possible role in tumour malignancy. Applying a machine learning tool known as the ‘Adaptive Index Model’ the combined expression of different CA12 isoforms, CD209 and CDH1 defined a ‘signature’ classifying HCC patients in groups at different risk of recurrence, thus indicating a link between pH regulators, myeloid and EMT markers likely influencing HCC prognosis. IHC analysis indicated that HCC displays a complex expression pattern for the pH regulatory proteins. Both CAIX and CAXII were detected in transformed, but not in normal hepatocytes. CAIX protein had a focal distribution in the tumour, thus supporting its possible association with hypoxic and the most aggressive tumour area. Conversely, CAXII was homogeneously expressed by all tumour hepatocytes, but mainly retained in the endoplasmic reticulum (ER). The majority of HCC expressed V-ATPase which, importantly, was also present in immune infiltrating cells. This expression pattern qualified the CAIX, CAXII and V-ATPase as possible targetable molecules. Our in vitro data indicated that blockage of their enzymatic activities by specific drugs affected the viability of HCC cell lines in a dose dependent fashion, although with the CAXII specific inhibitor showing low efficacy, likely related to the preferential ER localization of CAXII molecules inside the HCC cells. Ex vivo experiments with HCC tissue explants and HCC cellular suspensions showed that inhibition of VATPase modulated the epithelial/mesenchymal features of HCC cells and the levels of pro- and anti-tumour cytokines expressed by M2 macrophages and T cells infiltrating HCC.
Conclusions: Herein, our data demonstrated that the pH regulatory molecules, CAIX, CAXII and V-ATPase are over expressed in the HCC microenvironment and interfering with their pathways exerted anti-tumour activities, although these data also lead to the conclusion that more effective CAXII specific drugs should be designed. The results of this thesis also suggest that pH regulatory molecules might have a role in HCC aggressiveness and prognosis. Importantly, one of these pH regulators, namely V-ATPase complex, influences the mesenchymal features of tumour cells and the immunosuppressive tumour microenvironment (TME). Interfering with tumour metabolism is an emerging strategy for treating cancers that are resistant to standard therapies. Thus, targeting the unique crosstalk between tumour cells and the microenvironment, played by the pH regulatory molecules, can be considered as a new option for HCC treatment and the blockage of the V-ATPase complex might represent a multi-task strategy for the treatment of HCC patients
